Project Summary The goal of this work is to determine the three dimensional structure of the bacterial nucleoid and to gain insight into the mechanisms underlying the establishment and maintenance of this highly organized structure. The precise organization has recently been demonstrated in Caulobacter crescentus. In a non- replicating cell the position of a chromosomal locus within the cell correlates with its distance from the origin of replication. This order is maintained during replication, where newly replicated loci move rapidly and directly to the same relative position in the incipient daughter cell. To obtain a high resolution structure of the nucleoid, the three dimensional position of labeled chromosomal loci in the cell will be determined using electron microscopy and tomography. I will determine how this order is established by investigating the role of DMA binding factors known to affect chromosome structure. Analysis of the DMAcondensation ability of the Structural Maintenance of Chromosomes (SMC) protein will complement the genetic and cell biological studies of this protein. This work will lead to a new level of understanding of chromosomal structure and the mechanisms that couple chromosome replication, condensation and segregation. Relevance Due to the continued emergence of drug resistant bacteria, there is a rising need for novel targets for antibiotics. The success of existing antibiotics is based on their ability to disrupt essential processes within the bacterial cell. Maintenance of the proper organization of DMAin the cell is critical, thus understanding the mechanisms that govern the three-dimensional structure of the bacterial chromosome may lead to the identification of factors that are suitable targets for new antibiotics.